Background: Secondary-ion mass spectrometry (SIMS) is an important tool for investigating isotopic composition in the chemical and materials sciences, but its use in biology has been limited by technical considerations. Multi-isotope imaging mass spectrometry (MIMS), which combines a new generation of SIMS instrument with sophisticated ion optics, labeling with stable isotopes, and quantitative image-analysis software, was developed to study biological materials. Results: The new instrument allows the production of mass images of high lateral resolution (down to 33 nm), as well as the counting or imaging of several isotopes simultaneously. As MIMS can distinguish between ions of very similar mass, such as ^{12}C^{15}N^{-} and ^{13}C^{14}N^{-}, it enables the precise and reproducible measurement of isotope ratios, and thus of the levels of enrichment in specific isotopic labels, within volumes of less than a cubic micrometer. The sensitivity of MIMS is at least 1,000 times that of ^{14}C autoradiography. The depth resolution can be smaller than 1 nm because only a few atomic layers are needed to create an atomic mass image. We illustrate the use of MIMS to image unlabeled mammalian cultured cells and tissue sections; to analyze fatty-acid transport in adipocyte lipid droplets using ^{13}C-oleic acid; to examine nitrogen fixation in bacteria using ^{15}N gaseous nitrogen; to measure levels of protein renewal in the cochlea and in post-ischemic kidney cells using ^{15}N-leucine; to study DNA and RNA co-distribution and uridine incorporation in the nucleolus using ^{15}N-uridine and ^{81}Br of bromodeoxyuridine or ^{14}C-thymidine; to reveal domains in cultured endothelial cells using the native isotopes ^{12}C, ^{16}O, ^{14}N and ^{31}P; and to track a few ^{15}N-labeled donor spleen cells in the lymph nodes of the host mouse. Conclusion: MIMS makes it possible for the first time to both image and quantify molecules labeled with stable or radioactive isotopes within subcellular compartments.